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Puberty moderates genetic influences on disordered eating attitudes and behaviors, with little genetic influence before puberty but large (≥ 50%) genetic effects during and after puberty. To date, however, nothing is known about the mechanisms that underlie these effects. Estradiol is a particularly promising candidate, as estrogens become elevated at puberty and regulate gene transcription within neurotransmitter systems important for eating-related phenotypes. The aim of this pilot study was to examine whether estradiol levels moderate genetic influences on disordered eating during puberty.
Participants included 198 female twins (ages 10-15 years) from the Michigan State University Twin Registry. Disordered eating attitudes and behaviors were assessed with the total score, weight preoccupation, body dissatisfaction, and binge eating/compensatory behavior subscales of the Minnesota Eating Behavior Survey. Afternoon saliva samples were assayed for estradiol levels. Moderation of genetic effects was examined by comparing twin correlations in low versus high estradiol groups.
In the low estradiol group, monozygotic (MZ) and dizygotic (DZ) twin correlations for all MEBS scales were similar, suggesting little genetic influence. In the high estradiol group, the MZ twin correlation was more than double the DZ twin correlation, indicating the presence of genetic effects. Findings could not be accounted for by age, body mass index, or the physical changes of puberty.
Estradiol may be one important moderator of genetic effects on disordered eating during puberty. Larger twin studies are needed to replicate this pilot work and quantify the extent of genetic moderation.
Developmental twin studies suggest that puberty moderates genetic effects on disordered eating attitudes and behaviors (Klump et al., 2007a, Klump et al., 2003, Klump et al., 2007b). Using continuous measures of general disordered eating (i.e., sum scores of weight preoccupation, body dissatisfaction, binge eating, and the use of compensatory behaviors), little-to-no genetic influence was found for disordered eating in pre-pubertal twins (~0%), but significant genetic effects (~50%) were observed in twins who had begun puberty Follow-up studies have replicated these pubertal effects in two separate twin samples (Culbert et al., 2009, Klump et al., 2007b) and have shown that genetic effects emerge during early to mid-puberty rather than late puberty (Culbert et al., 2009). Overall, these findings are significant in identifying the differential influence of genes versus environment across development. Greater understanding of these developmental shifts may better inform prevention efforts by identifying developmental windows of increased risk and psychosocial and biological factors that are most relevant to the development of eating disorders.
Despite the potential significance of the work, little is known about the mechanisms underlying shifts in genetic effects. One of the primary hypotheses has centered on the initiation of ovarian hormone secretion during puberty (Culbert et al., 2009, Klump et al., 2003, Klump et al., 2007b). Ovarian hormones (estrogen, progesterone) become prominent in girls during puberty and have been found to predict changes in food intake in both animals and humans (e.g., (Asarian and Geary, 2006, Edler et al., 2007, Klump et al., 2008). Among ovarian hormones, estradiol is elevated early in puberty and circulating levels increase linearly throughout the pubertal period. This trajectory closely mimics the linear increases in genetic effects observed for disordered eating across puberty (i.e., 0-44%) (Klump et al., 2007b) and thus, increases in estradiol may account for increases in genetic effects during puberty. Estradiol is a steroid hormone that is known to be a potent regulator of gene transcription within the central nervous system (Ostlund et al., 2003a). The direct genomic effects of estradiol highlight its potential role as a moderator of genetic effects on disordered eating during puberty.
The purpose of this pilot study was to directly examine the effects of estradiol in a sample of same-sex female twins. We examined whether levels of estradiol moderate genetic influences on disordered eating during puberty, such that the heritability of these characteristics is greater in twins with higher estradiol levels as compared to twins with lower estradiol levels. We controlled for several potential confounding variables in analyses. Previous research has indicated significant age differences in genetic and environmental influences on disordered eating across adolescence using cross-sectional (Klump et al., in press, Klump et al., 2000) and longitudinal (Klump et al., 2007a) designs. In addition, body weight is influenced by estradiol levels (Asarian and Geary, 2006), tends to increase at puberty in girls (Grumbach and Styne, 1998), and is influenced by genetic factors (Allison et al., 1994). Finally, the physical changes of puberty are correlated with estradiol levels and have been proposed to contribute to increased mean levels of disordered eating (Slap et al., 1994). Given the potential importance of these variables, we controlled for age, body mass index, and the physical changes of puberty in analyses to ensure that they do not account for estradiol's moderation of genetic effects for disordered eating attitudes and behaviors.
Participants included 258 same-sex, adolescent female twins (129 pairs; 63 monozygotic (MZ), 66 dizygotic (DZ)) between the ages of 10 and 15 years (M = 11.98 years, SD = 1.40) drawn from the Michigan State University Twin Registry (MSUTR; (Klump and Burt, 2006). The MSUTR is a population-based twin registry that examines developmental differences in genetic and environmental influences for several forms of psychopathology. Recruitment methods for the MSUTR have been described in detail elsewhere (Klump and Burt, 2006). In brief, our adolescent sample was recruited entirely through birth records in Michigan in collaboration with the Michigan Department of Community Health (MDCH). The MDCH randomly selected same-sex female twins meeting our age requirements and living within 90 minutes of our MSU laboratories. Twins were recruited for study participation until our target sample size for this pilot project was reached. These recruitment methods result in MSUTR samples that are broadly representative of the population of Michigan (Culbert et al., 2008).
Zygosity was established through a physical similarity questionnaire (Peeters et al., 1998) completed by one parent (typically the mother). Indeterminable zygosity scores were resolved by a project Principle Investigator (KLK or SAB) through a review of item endorsements and/or twin photographs. These methods have been found to be over 95% accurate in determining twin zygosity (Peeters et al., 1998) and are used by many population-based twin registries (Iacono et al., 1999).
Twin age, twin ethnicity, and parental income were assessed using a standard demographic questionnaire completed by one parent.
Disordered eating was assessed using the Minnesota Eating Behaviors Survey (MEBS; (von Ranson et al., 2005). The MEBS was developed from the Eating Disorders Inventory (EDI) (Garner et al., 1983); item re-wordings and additions were made to make the measure appropriate for use with children as young as 9 years old (Klump et al., 2000). The MEBS has been employed in previous developmental twin studies of eating disorders (Culbert et al., 2008, Klump et al., 2007a, Klump et al., 2000, 2003, Klump et al., 2007b). Our use of the same, developmentally appropriate measure of disordered eating allows us to explore whether estradiol accounts for previously observed pubertal differences in genetic effects.
The MEBS includes 30 true/false items that assess overall levels of disordered eating via a total score as well as specific disordered symptoms using the weight preoccupation (8 items assessing preoccupation with dieting, weight, and the pursuit of thinness), body dissatisfaction (6 items assessing dissatisfaction with the size and/or shape of one's body), binge eating (7 items assessing the tendency to engage in binge eating and to think about binge eating), and compensatory behaviors (6 items assessing the tendency to use or to contemplate using inappropriate compensatory behaviors such as self-induced vomiting and laxatives to control weight) subscales. Psychometric properties of the MEBS total score, weight preoccupation, and body dissatisfaction subscales are excellent in pre- and early adolescent subjects. For example, the original factor analysis of the MEBS (see Klump et al., 2000) and follow-up analyses (see von Ranson et al., 2005) support factor congruence for these scales in pre-adolescent and adolescent girls (i.e., factor congruence coefficients > .88). These scales also exhibit good internal consistency (α's > .80 in the literature as well as the current sample), and they correlate highly (r ≥ .70) with similar measures of disordered eating (e.g., the Eating Disorder Examination Questionnaire) (von Ranson et al., 2005). Finally, all three scales successfully discriminate between adolescents with anorexia nervosa (AN) or bulimia nervosa (BN) versus control participants (von Ranson et al., 2005).
The MEBS binge eating and compensatory behavior subscales tend to be less internally consistent (i.e., α's < .65) in early adolescent girls (von Ranson et al., 2005). This was true in the current sample as well (α's < .65), particularly in the pre-adolescent girls. However, given that the original EDI from which the MEBS was developed includes items tapping binge eating and compensatory behaviors on the same bulimia scale (Garner et al., 1983), we explored whether a combined binge eating/compensatory behavior scale would exhibit acceptable internal consistency. The internal consistency of the combined scale was well within the acceptable range (α = .70). Consequently, in the current study, we focused our analyses on the MEBS total score, weight preoccupation, body dissatisfaction, and the combined binge eating/compensatory behavior subscale. This approach allowed us to examine a range of disordered eating characteristics and attitudes while maintaining the integrity of the twin analyses which tend to be quite sensitive to measurement issues and error (Plomin et al., 1990).
Salivary samples were used to obtain estradiol levels. Monitoring biomarkers in saliva has distinct advantages over doing so in other biological fluids (i.e., urine, serum, plasma) (Shirtcliff et al., 2000). For example, saliva sampling is a less invasive method that assesses the unbound hormone, which provides a more accurate indicator of active forms of estradiol. Saliva sampling also has been associated with higher compliance and more robust hormone-behavior associations than bloodspot samples (Edler et al., 2007).
Salivary samples were collected during the lab visit. Twins were asked to refrain from eating or drinking for four hours prior to the visit, and to refrain from smoking, brushing their teeth, or chewing gum for 30 minutes prior to the saliva collection. Twins then passively drooled down a straw into a cryovial until ≥ 4 ml was produced. All saliva collections occurred between 2:00 pm and 5:00 pm. We chose to collect during these times because afternoon-early evening diurnal variations in ovarian hormones during puberty tend to be minimal (Angold et al., 1999, Grumbach and Styne, 1998), and most families were available for assessments during this time.
Salivary specimens were frozen immediately and stored until they were shipped for analysis to Salimetrics, Inc. (Pennsylvania State University, College Park, Pennsylvania). On the day of testing, samples were centrifuged at 3,000 rpm for 10 minutes to remove mucins, and clear samples were pipetted into testing wells to screen for problems with pH. Samples testing outside of the pH range of 4-9 were diluted in PBS to correct pH prior to testing. Samples were then assayed in duplicate using an enzyme immunoassay developed by Salimetrics, Inc. The assay has a minimum detection limit of <1 pg/ml and average intra- and inter-assay coefficients of variation less than 7.13% and 7.45%, respectively. Method accuracy, determined by spike recovery and linearity, are 104.2% and 99.9% (acceptable ranges = 80-110%). Estradiol values from matched serum and saliva samples show a strong linear relationship for females (r=.80) (Shirtcliff et al., 2000).
Body mass index (BMI; weight (kg)/ height2 (meters2)) was calculated using laboratory measures of body weight and height. MSUTR research assistants measured twin weight using a digital scale and twin height using a wall-mounted ruler.
Physical changes of puberty were assessed using the Pubertal Development Scale (PDS; (Petersen et al., 1988). The PDS assesses growth spurts, skin changes, body hair growth, breast development, and the initiation of menses. Twins self-rated their development in these areas on a four-point scale: 1) development has not yet begun; 2) development has barely started; 3) development is definitely underway; and 4) development seems completed. Menstruation was rated dichotomously as absent (1) or present (4). Similar to previous research (Klump et al., 2003, Klump et al., 2007b, Petersen et al., 1988), average scores (i.e., range = 1-4) across all five items were used in analyses. Previous studies of the PDS psychometric properties have supported its reliability (median alpha = .77) (Petersen et al., 1988) and validity, including showing high correlations with clinician ratings of secondary sex characteristic development (r's = .61-.67) (Petersen et al., 1988).
The MEBS total scores were prorated for twins who were missing ≤10% of the items. Scores were coded as missing for twins missing more than 10% of items. Given the relatively small number of items on the weight preoccupation, body dissatisfaction, and binge eating/compensatory behavior subscales (range = 6-13 items), subscale scores of twins with one or more missing items were not prorated, but were instead coded as missing. Sample sizes therefore vary somewhat across subscale analyses. The MEBS subscale scores were log transformed to account for positive skew, which resulted in appropriate skew values between -1 and 1 for all scales. Finally, age, BMI, and PDS scores were regressed out of all MEBS subscale scores prior to analyses to ensure that estradiol's effects are not due to these potentially confounding variables.
Ideally, we would have examined differences in the heritability of these scores by estradiol levels using twin moderation models (Purcell, 2002). However, our sample size was too small for these analyses which typically require several hundred pairs to detect moderation (Purcell, 2002). Instead, in this preliminary study, we divided our twins into “low” versus “high” estradiol groups using the 50th percentile for estradiol levels (i.e., 5.79 pg/ml) and compared twin correlations in each group. The use of a 50th percentile split allowed us to maximize sample sizes in each group while still examining potentially meaningful differences in hormone effects.
Because MZ twins share approximately 100% of their segregating genes while DZ twins share, on average, 50%, significantly greater MZ relative to DZ twin correlations indicate the presence of genetic effects. By contrast, MZ and DZ twin correlations that are similar in magnitude and are significantly greater than 0 signify a lack of genetic effects, but significant shared environmental influences (i.e., environmental factors that are common to siblings growing up in the same family and contribute to their behavioral similarity). Finally, MZ twin correlations that are less than 1.00 indicate the presence of nonshared environmental factors (i.e., factors that are unique to siblings growing up in the same family and contribute to behavioral differences) and measurement error.
Importantly, because our analyses focus on comparing twin pairs with high versus low estradiol levels, we could only include pairs if the co-twins were concordant for either low or high estradiol status (Culbert et al., 2009, Klump et al., 2003). This resulted in the exclusion of 30 pairs from analyses (N = 14 MZ, 16 DZ) and a final sample of 99 pairs (49 MZ, 50 DZ). However, there were no significant mean or variance differences in MEBS total scores between twins from concordant versus discordant pairs (data not shown), suggesting that this exclusion did not unduly influence our results.
A range of disordered eating attitudes and behaviors was present in our sample. A total of 8.2% of twins scored above the clinical cut-off for the total score (cut-off score = 15.55) (von Ranson et al., 2005). Thus, our findings should speak to a variety of disordered eating attitudes and behaviors across the spectrum of severity.
Table 1 includes comparisons of demographic characteristics and study variables for the low versus high estradiol groups. There were no significant differences in ethnicity or parental income between groups (all p's > .05). However, and as would be expected given our categorizations, twins in the high estradiol group were significantly older, they were at later stages of pubertal development, and they exhibited higher mean levels of estradiol than twins in the low estradiol group. The two groups did not differ in BMI, mean levels or variability (p's > .05 for Levene's Test for Equality of Variances) of MEBS scores, or the proportion of twins scoring above the MEBS clinical cut-off for the MEBS total score (8.6% vs 9.6% in low and high estradiol groups, χ2 (1) = 0.22, p = .64). These findings confirm that differences in levels of disordered eating cannot account for potential differences in etiologic effects across groups. Importantly, however, the lack of differences in disordered eating by estradiol levels does not negate a moderating effect of estradiol on genetic effects, as the presence of genetic moderation would be expected to attenuate phenotypic associations. This pattern of results has been observed previously, where puberty was found to be a significant moderator of genetic effects despite negligible phenotypic associations with disordered eating (Culbert et al., 2009, Klump et al., 2003).
Twin correlations for the MEBS scales are depicted in Table 2. Differences between MZ and DZ twin correlations varied by estradiol status for all three MEBS scales. In the low estradiol group, the MZ and DZ twin correlations were approximately equal suggesting a lack of genetic effects. In these twins, it would appear that shared and nonshared environmental influences predominate. By contrast, in the high estradiol group, the MZ twin correlations were more than double the DZ twin correlations using all MEBS subscale scores. Notably, the MZ/DZ twin differences in the high estradiol group reached or approached traditional thresholds for statistical significance (i.e. p's = .03 -.12). Moreover, effect sizes for all twin correlations in the high estradiol group were in the moderate-to-large range (i.e., q's = .37-.59), suggesting clinically significant differences in MZ and DZ twin similarity for disordered eating.
Nonetheless, we were interested in exploring whether statistically significant differences in twin correlations would emerge in the high estradiol group if we examined a more extreme group. We consequently re-constituted our high estradiol twin group into those falling in the highest tertile of estradiol levels (estradiol level = 8.13 pg/ml; n = 29 pairs, 14 MZ, 15 DZ). The difference in twin correlations now reached statistical significance for total score (rMZ =.88; rDZ =.17; Z = 2.88, p = .002), weight preoccupation (rMZ =.81; rDZ = - .06; Z = 3.44, p = .0003), and body dissatisfaction (rMZ =.69; rDZ = - .14; Z = 2.37, p = .008), and approached significance for the binge eating/compensatory behaviors subscale (rMZ =.78; rDZ = .34; Z = 1.40, p = .08). Taken together then, findings suggest the presence of genetic effects on disordered eating in the high estradiol group. They further indicate a lack of shared environmental influence, but significant nonshared environmental effects, on disordered eating at high estradiol levels.1
Findings provide preliminary support for our hypothesis that levels of estradiol moderate genetic effects on several types of disordered eating attitudes and behaviors. Using a pilot sample of twins, disordered eating exhibited little-to-no genetic influence in twins with low estradiol levels during puberty, but moderate-to-substantial genetic effects in twins with higher estradiol levels. These effects were present even when controlling for age, BMI, and the physical changes of puberty. Taken together, results provide initial support for a role for estradiol in the genetic diathesis of disordered eating attitudes and behaviors during puberty.
The unique effects of estradiol levels above and beyond the influence of the confounding variables deserves note. These findings suggest that estradiol's effects are unlikely to be artifacts of age, increased body weight, or the physical changes during puberty that have been hypothesized to influence disordered eating risk in girls. Results also indicate that age differences in genetic effects observed previously (i.e., increases in heritability between ages 11 and 14; no changes in heritability from age 14 and on) (Klump et al., in press, Klump et al., 2000, Klump et al., 2007b) may be due to increases in estradiol that characterize puberty and typically occur between the ages of 11 and 14 years (Kaltiala-Heino et al., 2003).
Moving forward, it will be important to replicate these findings using larger sample sizes and explore mechanisms underlying estradiol's effects. A critical function of estradiol during development is the regulation of gene transcription within the central nervous system (CNS) (Ostlund et al., 2003b). When activated by hormone, intracellular steroid hormone receptors act as transcription factors to regulate mRNA expression in neurobiological systems. Increased genetic effects on disordered eating during puberty may therefore result from increases in estradiol and its genomic effects on the structure and/or function of the CNS through the production of neurotransmitters, their receptors, or their signal transduction mechanisms. Animal data show that genes and gonadal hormones influence the pattern and timing of changes in brain structure and function during puberty (Ahmed et al., 2008, Nunez et al., 2002, Primus and Kellogg, 1991, Spear, 2000, Zehr et al., 2006). Emerging data suggest that these same effects may be present in humans, as levels of estradiol are inversely correlated with changes in neural structures in girls during puberty, and these neural changes appear to be influenced by genetic factors (Lenroot et al., 2009, Peper et al., 2009a, Peper et al., 2009b). Taken together, data across disparate sources suggest that increases in estradiol during puberty may increase genetic influences on disordered eating through differential organization of neural circuitry underlying risk.
Notably, our findings may appear to conflict with those showing that lower levels of estradiol are associated with increased levels of binge eating over the course of the menstrual cycle in women with BN (Edler et al., 2007) and women from the community (Klump et al., 2008). However, the current study focused on a different level of analysis (i.e., within-twin pair similarity versus within-person associations) than previous work. Thus, the current work examines the potential influence of estradiol on heritability of disordered eating over puberty whereas previous work has examined the potential influence of estradiol on levels of disordered eating in adult women. The effects of estradiol on disordered eating may differ by developmental stage, such that inverse phenotypic associations are observed in adulthood, whereas no phenotypic (but significant genetic) associations are present during puberty (Klump and Keel, in preparation). These differential effects fit nicely with the organizational/activational hypothesis of gonadal hormone action which predict: 1) gonadal hormones organize risk for disordered during puberty through changes in gene transcription and resulting changes in brain structure/function; and 2) these changes organize the brain to respond to circulating levels of hormones in adulthood which activate and/or influence the expression of behavior (Sisk and Zehr, 2005). When hormone organization/activational effects are present, modest phenotypic associations between gonadal hormones and behavior are observed during puberty, whereas significant phenotypic associations are present in adulthood (Sisk and Zehr, 2005). This pattern of phenotypic associations fits nicely with that observed for disordered eating and could help explain seemingly disparate findings for estradiol during puberty versus adulthood (Klump and Keel, in preparation).
Much more research is needed to confirm and replicate our pilot study results and examine speculative hypotheses regarding mechanisms of estradiol's effects. Ideally, future work would address limitations of our pilot study. Our sample sizes were small and limited our ability to conduct biometric model-fitting to quantify the degree of genetic moderation. We examined a community sample of twins whose findings may not generalize to a clinically diagnosed sample. Given the very low prevalence of AN and BN in pre- and early adolescence (Bulik, 2002), it would be difficult if not impossible to examine our hypotheses in a purely clinical sample of twins. Our sample exhibited a wide range of disordered eating attitudes and behaviors, including clinical levels of these characteristics. Thus, our findings likely speak to the range of disordered eating present in the population at large. Nonetheless, additional work in larger twin samples is needed to determine the generalizability of the findings to AN and BN. Studies in larger sample also may track estradiol's role in moderating genetic effects on disordered eating in specific age groups.
Finally, we collected salivary hormone and disordered eating data on a single day. It is preferable to obtain multiple measures on different days to obtain more stable estimates of these phenotypes, particularly estimates of estradiol levels. Future research should obtain multiple measures of hormones, eating disorder phenotypes (i.e., self-report, interview), and covariates in larger samples of twins to confirm and extend this pilot work.
Data collection for the research presented herein were supported by grants from the National Institute of Mental Health awarded to Dr. Klump (MH 070542) and by grants from the Intramural Research Grants Program at Michigan State University awarded to Drs. Burt (04-232) and Klump (71-4831). The authors would like to thank the MSUTR twins for their participation in this research and the MSUTR staff for their help in collecting and managing the study data.
1Because some of our twins were post-menses at the time of study, we re-ran all of our correlations excluding twins who had experienced at least one menstrual cycle (N = 90 twins). Despite the large decrease in sample size, the pattern of results remained unchanged, with minimal genetic effects in the low estradiol group for total score (rMZ =.59; rDZ = .46; Z = 0.38, p = .35), weight preoccupation (rMZ =.41; rDZ = .49; Z = -0.21, p = .42), body dissatisfaction (rMZ =.54; rDZ = .34; Z = 0.51, p = .31), and binge eating/compensatory behaviors (rMZ = -.09; rDZ = -.01; Z = -0.16, p = .44), but indications of genetic effects in the high estradiol group, where the MZ twin correlation was more than double the DZ twin correlation for total score (rMZ =.34; rDZ = .06; Z= 0.53, p = .29), weight preoccupation (rMZ =.48; rDZ = .19; Z = 0.68, p = .24), body dissatisfaction (rMZ =.41; rDZ = .11; Z = 0.38, p = .35), and binge eating/compensatory behavior (rMZ =.41; rDZ = .15; Z = 0.49, p = .31) subscales.